The long-term aim of this work is to understand the underlying molecular mechanisms by which naturally occurring stimuli regulate the opening and closing of the BK-type calcium (Ca2+)-activated potassium (K+). A common characteristic shared by BK channels with essentially all other ion channels and also many other categories of protein is that sensing of a physiological stimulus on one part of the protein is coupled to regulation of a key functional property occurring on another part of that protein. In the case of BK channels, sensing of either changes in membrane voltage or changes in cytosolic Ca2+ regulate the activation of ion flux through the channel. The fact that BK channels respond independently to two distinct physiological signals has been an advantage for investigation of the underlying molecular steps that link these processes. This project will focus specifically on two major aspects of how activation of BK channels is regulated. First, the role of conformational changes in the selectivity filter region of the channel in channel activation will be examined. Here, how selectivity filter gating is coupled to either voltage changes or Ca2+ elevations will be examined. Second, the structural features of BK channels that influence coupling between ligand binding and channel activation will be examined. Using methods of electrophysiology combined with molecular biology, this project will take advantage of two closely related ion channels, the BK channel and its pH-regulated homologue, Slo3. Important functional differences between these two channels can be exploited to map the loci responsible for the functional difference. Coupled with mechanistic analyses, information regarding the coupling between gating and ligand dependence can be defined. Together these questions address important general issues that are likely to have broad significance to a wide variety of ion channels. BK channels are of broad importance in the normal functioning of a variety of excitable cells. By responding to both elevations in cytosolic Ca2+ and changes in membrane potentials, BK channels play an important role in the regulation of cellular excitability. Among different tissues, BK channels contribute to regulation of neuronal excitability, smooth muscle relaxation, synaptic transmission and hormone release. Better understanding of the regulation of BK channel function is of potential medical importance, not only because the channels may serve as specific therapeutic targets but also because altered function of particular variants may contribute to unknown pathological conditions.